The invention relates to a method for the analysis of a layer of solution adjacent a surface of an electrode.
Electrochemistry has for a long period been extensively used for the analysis of solutions, including analysis of the generation of reactive species in solution, usually on a time scale of a few tens of seconds down to the sub-millisecond range. Most electrochemical analysis is effected with controlled potential experiments, such as voltammetry and chronoamperometry, in which a controlled potential is applied to the electrode and the current passing through the electrode is monitored and used to deduce mechanistic and thermodynamic data as well as concentrations. While such methods dependent upon current measurement have yielded much useful analytical data, such methods often suffer from an inherent lack of selectivity. Voltammetry is a low-resolution method, making it difficult to measure a single trace component in the presence of other species. Faradaic reactions of adsorbed molecules or the electrode itself may generate significant "noise" signals, making it impossible to detect very small concentrations of solution components in many cases. In addition, double layer chrging current is always present in voltammetry, and this double-layer charging current also serves to lower the sensitivity to components present in only small amounts. Moreover, in voltammetric experiments, the observed current may be due to reactions of several different species in the solution and, if the chemical changes involved are complex, the mere observation of the overall current is often insufficient to enable one to deduce the mechanism of the chemical changes taking place, or even to measure an overall rate constant for the chemical change.
Thus, while voltammetric techniques are theoretically applicable to any componet of a solution which can undergo oxidation or reduction, the aforementioned problems severely limit the usefulness of the technique for analysis of complex organic mixtures such as biological fluids. The limitations of voltammetric techniques with biological fluids are still severe, despite the recent development of pulse techniques which allow analysis down to the 10.sup.-8 to 10.sup.-9 M concentration range in a few cases. Accordingly, although voltammetric techniques are used in certain instances, in most cases the aforementioned difficulties have prevented the application of voltammetric techniques to biological fluids without major sample preparation.
Recently so-called "spectroelectrochemical" methods have been developed which greatly increase the selectivity of electrochemical analytical methods. In spectroelectrochemical methods, electromagnetic radiation (usually either visible or ultraviolet) is passed through the layer of solution adjacent the electrode at which the electrochemical changes are taking place. The species present in this layer of solution will of course produce conventional absorption spectra, so that if a chromophore (a term which is used herein to mean a species which absorbs certain wave lengths of electromagnetic radiation, not necessarily in the visible spectrum) is generated or consumed in the electrochemical reactions taking place at the electrode, the progress of the reactions and the concentrations of the species involved may be determined by inspection of the absorption spectra produced. A variety of arrangements have been used for passing the beam of radiation through the layer of solution adjacent an electrode. Most of these arrangements involve the use of an optically transparent electrode either in the form of a transparent substance such as glass coated with a very thin, transparent layer of a conductive metal, or in the form of a metal grid having apertures therethrough. For example, a grid-type optically transparent electrode may be positioned in a thin layer (typically about 0.2 mm thick) of solution sandwiched between two transparent plates. A beam of light is then shone through this apparatus, which functions as a small transmission cell in exactly the same manner as the much thicker cells used in conventional spectrophotometers. Absorption spectra may be obtained from the metal-on-glass type of optically transparent electrode either by passing a beam of light through the electrode or by arranging for total internal reflection of light from the surface of the electrode. Finally, absorption spectra may be obtained by bouncing a beam of light off a polished electrode (which need not be of the optically transparent type).
It is also possible to generate a Raman spectrum of a layer of solution adjacent an electrode by bouncing an intense beam of light (usually from a laser) off the surface of an electrode and examining the Raman-scattered light.
A review of the aforementioned spectroelectrochemical techniques may be found in W. R. Heineman, Analytical Chemistry, 50, 390A (1978).
Unfortunately, all the techniques described above are very insensitive since the path length for absorbance by chromophores generated at the electrode is limited to the thickness of the electrochemical diffusion layer, which is typically about 0.1 mm. In the case of total internal reflection, the path length is even less since the internally reflected beam only penetrates on the order of a few micrometers into the solution layer. These very short path lengths render the techniques relatively insensitive; even when strong chromophores are being generated, the methods are not generally useful at chromophore concentrations below 10.sup.-5 M, unless impractically long (about 100 second) electrolysis times are used. Moreover, because it is necessary to generate high concentrations of chromophores and establish a relatively thick diffusion layer of electrogenerated species in order to obtain sufficient absorbtion, spectroelectrochemical methods have not hitherto been generally usable when it is desired to study short-lived intermediates.
J. F. Tyson and T. S. West in Talanta 26, 117-125 (1979) and 27, 335-342 (1980) describe a spectroelectrochemical analytical method in which a light beam passes at grazing incidence over a platinum electrode. Electrogenerated chromophores in the solution layer adjacent the electrode selectivety absorb light from the beam and the beam is monitored after it has passed the electrode to determine the absorption occurring. Although this method is capable of improving the sensitivity of spectroelectrochemical measurements to some extent, it is difficult to control the grazing incidence of the beam sufficiently to obtain highly reproducible results and the method still demands the formation of a thick diffusion layer adjacent the electrode, so that electrolysis must be continued for a relatively long period, typically about 40 seconds. This renders the method useless for very short-lived species and prevents the method being used with modulation of the potential applied to the electrode. The use of such modulation is highly desirable because it can be used to improve greatly the sensitivity of the method.
There is thus a need for a spectroelectrochemical analytical method which does not require the formation of a thick diffusion layer and which thus permits the modulation of the voltage applied to the electrode in order to increase the sensitivity of the method. It is also desirable that such a method not be limited to an absorption path only equal to the thickness of the diffusion layer adjacent the electrode. This invention provides such a method.